First molecular detection of Neospora caninum from naturally infected slaughtered camels in Tunisia

Abstract Background Neospora caninum has been documented to infect most domestic wildlife but is known to primarily infect dogs and cattle and is considered an important cause of abortion in camels. Objective The aim of this study was to estimate the molecular detection of Neospora caninum in tissues of naturally infected camelids. Methods Brain, tongue (bottom and tip) and masseter muscles from 35 slaughtered camelids from Tataouine and Médenine regions were collected (n = 140 samples). PCR was used to amplify and detect N. caninum DNA in tissues samples followed by sequencing of some PCR products. A phylogenetic tree was then constructed to compare the partial sequences of the ITS1 gene with GenBank sequences. Histopathology examination was used to detect Neospora spp. cysts, but no lesions were observed. Results The overall molecular detection of N. caninum in camelids was 34.3% (12/35). The highest molecular detection of N. caninum was recorded in animals of more than 3 years old (6/9) and in animals aged between 1 and 3 years old (4/12). Whilst, the lowest molecular detection (2/14) was observed in animals 1 year or younger (p = 0.035). There were no significant differences in molecular detection of N. caninum according to both locality and gender (p > 0.05). Similarly, there was no difference of prevalence between different anatomical locations. Comparison of the partial sequences of the ITS1 gene revealed 100–95.5% similarity among our N. caninum amplicon (MW551566) and those deposited in GenBank. Conclusion These results highlight the presence of a risk infection by N. caninum in camels. For preventing N. caninum infection further studies are needed to improve our knowledge about the epidemiology of neosporosis in North Africa.

As far as we know, there are no data about the molecular detection of N. caninum in camels in Tunisia. The present study is the first to detect of N. caninum DNA in Tunisian dromedary camels slaughtered in the regional slaughterhouse of Médenine and Tataouine (Southern Tunisia).

Study area and specimen collections
Tissue samples were collected from 35 camels presented for slaughter at the regional slaughterhouses of two governorates from south Tunisia: districts of Médenine and Tataouine characterized by arid and Saharan climate, respectively ( Figure 1; Table 1). In Médenine district, the mean temperature in winter and summer is 12 and 27.7 • C, respectively. The mean annual rainfall is 156 mm with very large interannual variations. In Tataouine district, the mean temperature in winter and summer is 11.2 and 27.7 • C, respectively. The mean annual rainfall is 134 mm with very large interannual variations. The camel population in the two governorates included in the present study is estimated to be 12,293 and 11,000, respectively. http://www.ods.nat.tn/fr/index.php? id=32 One hundred and forty tissue samples were collected from the bottom and tip of the tongue, masseter muscle and brain of 35 slaughtered dromedaries belonging to different age groups and both genders.
Samples were collected in sterile and identified bags, transferred to the laboratory of parasitology, National School of Veterinary Medicine of Sidi Thabet, Tunisia and stored at −20 • C until processed.

DNA extraction
After thawing the samples, for each tissue sample location, 50 mg of tissue was cut using a sterile disposable blade for DNA extraction. Each tissue sample was washed with sterile distilled water and centrifuged at 16,000 g for 6 min. DNA was extracted using Wizard Genomic DNA purification kit (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions then stored at −20 • C until analysed.
To appreciate the quality of DNA in each extract prior to PCRs, universal PCR was performed for each sample using forward and reverse primers 1A and 564R (

PCR amplification of the ITS1 gene of N. caninum
A nested PCR was performed with four oligonucleotides to amplify a 279 bp N. caninum DNA fragment belonging to ITS1 gene and coding for the 18S-5.8S rRNA according to the protocol of Buxton et al. (1998).
A primary PCR was performed with 0.15 µM of each primer (NN1 and NN2) (Table S1) in a total reaction volume of 25 µL consisting of 3 µL of DNA sample, 1x PCR buffer, 2.5 mM MgCl 2 , 200 µM dNTP each, 1 U of Taq Polymerase. The amplification was carried out in a thermocycler under the following cycling conditions: 95 • C for 5 min, followed by 26 cycles (denaturation at 94 • C, annealing at 48 • C and extension at 72 • C for 1 min each) and a final extension at 72 • C for 5 min. We added 2 µL of the amplicons as template for the second PCR using the same mixture as primary PCR and 0.2 µM of each inner primer NP1 and NP2 (Table 1)    Local Alignment Search Tool (BLAST). MEGA 5 software was used as described by Tamura et al. (2011), to perform multiple sequence alignments. A phylogenetic tree was constructed using the Neighbourjoining (NJ) algorithm (Saitou & Nei, 1987) as implemented in MEGA 7

Histopathological examination
Small tissue samples (0.5 × 0.5 cm) from the tongue, masseter muscle and brain were collected and fixed in 10% neutral buffered formalin for 48 h. They were dehydrated in graded alcohol series, cleared in toluene then processed by the standard paraffin embedding technique.
The slices were cut at 4 µm thick, and mounted on microscope slides.
They were stained with hematoxylin and eosin (HE) and finally examined under an optical microscope at 400× then 1000× magnifications for the detection of Neospora spp. cysts.

Statistical analysis
Descriptive statistics, including molecular prevalence of N. caninum infection in each location, age group and gender were estimated. One dromedary was considered N. caninum-infected if at least one of the samples was PCR positive. Differences in molecular prevalence for all parameters were analysed by chi square Mantel-Haenszel test with Epi Info 6 software at 5% threshold (Schwartz, 1993). Odds ratios were estimated in each animal group (Ancelle, 2006).

Molecular detection of N. caninum
All tested samples were positive for 18S rRNA universal PCR. Out of 35 tested animals, 12 were N. caninum PCR-positive corresponding to an overall molecular detection of 34.3%.

F I G U R E 2 Venn diagram of interaction between Neospora caninum molecular infection prevalence in different categories samples
The highest molecular detection of N. caninum was recorded in animals of more than 3 years old (6/9) and in animals aged between 1 and 3 years old (4/12), whereas the lowest molecular detection (2/14) was observed in animals 1 year or younger (p = 0.035).
There were no significant differences of N. caninum molecular detection according to both locality and gender (p > 0.05). Similarly, there was no difference of prevalence between different anatomical locations ( Figure 2) (Tables 2 and 3).

Histopathology
Neither microscopic lesions nor N. caninum cysts were observed in all tissue-stained samples examined under microscope.

DISCUSSION
Neospora caninum has been documented to infect most domestic wildlife but is known from primarily infect dogs and cattle and is considered an important cause of abortion in camels (Hosseininejad et al., 2009;Wolf et al., 2005).
Little is known about the presence of N. caninum in meat tissues from naturally infected camels in North Africa. To our knowledge, this is the first molecular report of N. caninum in Tunisian slaughtered camels.
In Southern Tunisia, camels are an important source of meat, leather and labour. They are also important source of wealth and social status. Studies detecting N. caninum DNA in Tunisian have been conducted in naturally infected goats, sheep and cattle (Amdouni et al., 2018a(Amdouni et al., , 2018b(Amdouni et al., , 2019 but there are no data about Tunisian camels. Anti-N. caninum antibodies were detected in various ruminants' species worldwide. Seroprevalence reaches 87% in cattle, it ranges from 0 to 64% in sheep, 0 to 26.6% in goats and 3.7 to 86% in camels Selim & Abdelhady, 2020). For camels, seroprevalence was varied between 11.1 and 86% (Bártová et al., 2017;Hamidinejat et al., 2013;Hilali et al., 1998;Mentaberre et al., 2013;Mohammed et al., 2020;Nazir et al., 2017;Sadrebazzaz et al., 2006;Selim and Abdelhady, 2020). The highest prevalence of anti-N. caninum antibodies was reported in Canary Islands (86%) by Mentaberre et al. The molecular detection of N. caninum in camels reported in the study herein was greater than in Egypt at 24% (12/50) (Ahmed et al., 2017). Such differences of molecular detection can be attributed to differences in farming practices, hygienic measures, geographic factors and environmental influences Selim & Abdelhady, 2020).
Some findings have shown that N. caninum infection was positively correlated with age of animals and prevalence increase significantly in adult animals (Iovu et al., 2012;Nazir et al., 2017;Selim & Abdelhady, 2020). We found the same trend herein, the highest N. caninum molecular detection was found in animals of more than 3 years old (6/9) and in animals aged between more than 1 and 3 years old (4/12). Whilst, the lowest molecular detection (2/14) was observed in animals aged of 1 year or less (p = 0.035). This association with age can be explained by the repetitive cumulative infections and indicates the role of horizontal transmission in infection transmission (Selim & Abdelhady, 2020).